We also analyzed the group mean rejection rates as a function of the morph level and fitted with a cumulative normal distribution function. Results showed that the adults and the oldest 11-12-year-old children exhibited a greater sensitivity (d’) and a smaller discrimination threshold (μ) in the Asian-parent condition than those in the Caucasian-parent condition, indicating the presence of an own-race advantage. On the contrary, 5- to 10-year-old children showed an equal sensitivity and similar discrimination thresholds for both conditions, indicating an absence of the own-race advantage. Moreover, a gradual development in enhancing the discriminability for the Asian-parent condition was observed from age 5 to 12; however, the progression in the Caucasian-parent condition was less apparent. In sum, our findings suggest that expertise in face processing may take the entire childhood to develop, and supports the perceptual learning view of the other-race effect—the own-race advantage seen in adulthood likely reflects a result of prolonged learning specific to faces most commonly seen in one’s visual environment such as own-race faces.Previous studies examining the other-race effect in school-age children mostly focused on recognition memory performance. Here we investigated perceptual discriminability for Asian-like versus Caucasian-like morph faces in school-age Taiwanese children and adults. One-hundred-and-two 5- to 12-year-old children and twenty-three adults performed a sequential same/different face matching task, where they viewed an Asian- or a Caucasian-parent face followed by either the same parent face or a different morphed face  and judged if the two faces looked the same. We computed the  The human face carries abundant visual information and social cues. In daily encounters, we automatically attend to people’s gender, age, race, and facial expression, and these characteristics may well influence our social evaluations. For example, adults belonging to one racial group typically find it difficult to recognize or memorize faces of other racial or ethnic groups than those of their own . This phIn literature, the ORE or ORA has been demonstrated mostly as a bias in recognition memory where people can better retrieve own-race faces than other-race faces over certain retention intervals; this is particularly true for the developmental ORE literature in children. However, the existence of a recognition memory bias does not exclude the possibility that an own-race advantage may exist in the earlier perceptual encoding stage. In line with the broadly-defined perceptual expertise or learning view of the ORE (for a review see Meissner & Brigham ), indiviWhen does such a bias for own-race faces emerge in life? In the last decade, convergent evidence from cross-cultural studies has pointed to an early onset of the ORE in the first year of life. Studies based on preferential looking methods revealed that 3-mo-old Caucasian infants (but not newborns) exhibited a spontaneous looking preference for the Caucasian face when paired with other-race faces . LikewisSince the ORE has an early inception in the first year, one may wonder whether a continuous developmental trajectory of the ORE exists from infancy to childhood. Although the empirical research with infants and toddlers aged between 1 and 3 is lacking, several studies explored the other-race effect in preschoolers, children, and teenagers. Up to date, two studies reported the presence of the other-race effect in 3 year-olds. Using a simultaneous matching task, Sangrigoli et al. tested 3-, 4- and 5-year-old Caucasian children’s face recognition by asking them either pointing to the target face  or making a key press  . ResultsThe onset of a clear-cut ORE in childhood remains inconclusive. Pezdek et al. tested 5-, 8-year-olds and adults of African American or European American descent with a 6-person line-up recognition memory task one day later . They foConsistent with Goodman et al., two stunon-equal physical similarities between the selected faces from two different races. For example, it is not impossible that, in one study, young Caucasian children showed better recognition for Caucasian faces might be due to the selected Caucasian faces were more dissimilar than the Asian ones. But in another study where the physical similarity between the chosen race faces was equated, children in that study did not exhibit a reliable ORE at the same developmental age.To sum up, previous studies on the development of ORE in children mostly relied on the standard old/new recognition memory task. While the own-race advantage is stable in adulthood, the inconsistent results in children suggested that the own-race advantage may be highly plastic in childhood and particularly malleable in infancy and preschool age. Methodologically, the contradictory findings could reflect various procedural discrepancies among studies, such as the length of memory retention intervals, or the encoding time in the learning phase. Another major confounding factor could be the In light of this, the present study employed the morphing face paradigm to examine the encoding advantage hypothesis in Taiwanese school-aged children and adults as a comparison. We aimed to characterize the development of the other-race effect in children aged between 5 and 12 with a sequential same/different face matching task, which put more weight on perceptual discriminability than memory retention or retrieval capacity. We adopted the morphing paradigm with cropped female faces for several methodological considerations. First, the morphing technique allows for better control over the physical similarity between two selected faces. Secondly, we eliminated the external facial cues to encourage the participants to focus on the internal features and the configural information within the face. Lastly, the morphing technique created a linear continuum of very subtle changes suitable for estimating perceptual discriminability at near threshold level. In other words, we can calculate the minimum physical difference needed to produce a detectable perceptual change  with curve fitting procedures, and hence to chart the developmental progression of the perceptual discriminability for own- and other-race faces in children aged 5 to 12 and young adults.The adult participants and the parents of the child participants were informed of the general purpose and the experimental procedures of the study. Written consent  of the participants were obtained before the experiment. The protocol of the present study was approved under the Clinical Trial/Human Research Approval by the Research Ethics Committee, China Medical University and Hospital, Taichung, Taiwan. .A total of 23 adults  participated the experiment. The majority of the subjects were undergraduate and graduate students recruited from China Medical University via campus-wide advertisement. Informed written consent was obtained before the experiment. All participants were native Chinese speakers and had a normal or corrected-to-normal vision (20/20). None of the participants had the experience of living in countries where Caucasian is the majority race more than six months. The observers were naïve to the purpose of the experiment and were tested individually in a quiet, dimly lit room. Each participant received both the Asian- and Caucasian-parent conditions, and the test order was counterbalanced among participants. Each participant received cash compensation at the end of the experiment.A desktop computer (Acer Veriton M460) with 22” LCD monitor (Chimei CMV 221) and E-Prime Professional 2.0 software  were used to run the experiment. We adopted three Asian and three Caucasian female faces as the stimuli. The Asian faces were selected from the Taiwanese Facial Expression Image Database, TFEID , while tThe six female faces formed three pairs , we then used FantaMorph 5 Deluxe (Abrosoft Co.) to create three sets of morphed images on a linear continuum between the paired faces. A total of 65 key points for the facial features were kept constant for each of the oval-cropped Asian- and Caucasian-parent faces, which included 11 points on the mouth, 6 points on each eye, 10 points on the nose, 4 points on each eyebrow, and 24 points for the contour of the face. The FantaMorph program generated control points for a series of morph faces by moving 15%, 30%, 45%, and 60% of the total distance along the vector that connected corresponding control points in the Asian-parent and Caucasian-parent faces. As shown in The Adult Experiment incorporated two independent within-subject factors: the race condition (Asian-parent vs. Caucasian-parent) and the morph level of the face stimuli . The experiment contained two race blocks, and the test order was counterbalanced among participants. Each block included 72 trials presented in random order . A total of 107 participants , aged between 4.9 and 13.4 years, participated the study. The majority of the children were recruited from the Taichung Metropolitan Area via a parenting club on Facebook and Internet advertisements. Informed parental consent was obtained before the experiment. All child participants were native Chinese speakers and had a normal or corrected-to-normal vision (20/20). None of the child participants had the experience of living in countries where Caucasian is the majority race by their parent’s report. The child participants were tested individually in a quiet, dimly lit room. Each participant received both the Asian-parent and Caucasian-parent conditions, and the test order was counterbalanced among participants.The participants were divided into four age groups: 5- and 6-year-olds, 7- and 8-year-olds, 9- and 10-year-olds, and 11- and 12-year-olds. In the youngest 5- and 6-year-old group, 28 children were tested, two of them were excluded from the final data due to procedural errors (1) and a response bias (1) . The final sample size was 26 , with a mean age of 6.17 years (range between 4.99 and 6.97 years old). In the 7- and 8-year-old group, 30 children were tested, but three of them were excluded from the final data due to procedural errors (1) and a high false alarm rate (2)  being responded as “different”). The final sample size was 27  with a mean age of 7.96 years (range between 7.00 and 8.99 years). In the 9- and 10-year-old group, 28 children  completed the study, and all of them retained in the final data set. Their mean age was 9.83 years (range between 9.00 and 10.86 years). In the eldest 11- and 12-year-old group, 21 children  were tested and retained in the final data set. Their mean age was 11.55 years (range between 11.00 and 13.04 years old).The apparatus and the face stimuli were the same as in the Adults’ Study.The Children’s Study adopted a mixed design. The between-subject factor was age groups , while the within-subject factors were race (Asian-parent vs. Caucasian-parent) and the morph level of the face stimuli . The procedure of the sequential face matching task was the same as in the adult study. Likewise, the dependent variable was the “rejection rate,” defined as the percentage of judging the comparison face as “different”.hit, false alarm, miss, and correct rejections), and compute d’ as an index of sensitivity based on the hit rates and false alarms. For a given age group, a higher mean d’ in the Asian-parent condition than that in the Caucasian-parent condition suggests an own-race encoding advantage. Moreover, an interaction between age and race would signal a developmental change in the own-race advantage. This set of data are shown on the right side of To fully capture the characteristics and developmental changes in perceptual sensitivity for the Asian-parent and the Caucasian-parent conditions, we employed three data analysis methods applying to all age groups. First, as half of the trials were physically the same  and half were physically different , we adopted signal detection theory to code the participant’s four response categories   and the group slope parameter (σ)  of the psychometric functions in children and adults. Therefore, for a given age group, the presence of a smaller discrimination threshold (or a sharper slope) in the Asian-parent condition than that in the Caucasian-parent condition would indicate the presence of an own-race advantage. The curve fitting data are summarized numerically in Last but not least, the morph level of the face stimuli can also be regarded as a continuous variable, and the performance dimension  increases monotonically with the stimulus intensity . Thus, we further fitted the group mean psychometric functions  with a hits” and the “correct rejections.” Judging the comparison face as “different” on the physically “different” trials was denoted as “hit” . Judging the comparison face to be the “same” on the physically same trials was denoted as “correct rejections.” Similarly, there were two types of incorrect responses, the “miss” and the “false alarm.” Judging the comparison face to be “the same as the target face” was denoted as “miss” for the physically “different” trials . Lastly, judging the comparison face to be “different” on the physically “same” trials where the two face images were identical was denoted as “false alarm”.In the current same/different face matching task, half of the trials were physically the same  and half were physically different . The participants would yield four possible response categories according to the Signal Detection Theory (SDT) , 38. Conhits, false alarms, misses, correction rejections, and d’s for the Asian-parent and the Caucasian-parent conditions separately. A two-way mixed ANOVA was performed on the d’s with Age group  as the between-subject factor, and Race (Asian-parent vs. Caucasian-parent) as the within-subject factor. The Age group main effect was significant F = 10.496, p < .001, η2 p = .259; the group mean d’s increased with age which were 0.952 (SE = .103), 1.184 (SE = .101), 1.242 (SE = .097), 1.504 (SE = .114), and 1.834 (SE = .109) for the 5–6 yr olds, 7–8 yr olds, 9–10 yr olds, 11–12 yr olds, and young adults, respectively. The Race main effect was also significant F = 13.899, p < .001, η2 p = .104; the mean d’ was greater for the Asian-parent condition  than that for the Caucasian-parent condition .The sensitivity index of d', defined as the Z(Hit)-Z, for the Asian-parent and Caucasian-parent conditions were computed separately. This sensitivity index tells us how well can adults and children distinguish between the briefly presented target faces and the following comparison faces. Age group * Race interaction was significant F = 3.402, p = .011, η2 p = .102, indicating that the difference in the perceptual sensitivity for Asian versus Caucasian condition varies with age. To reveal whether the participants of certain age group exhibited an own-race encoding advantage—a higher mean d’ in the Asian-parent than that in the Caucasian-parent condition, we further analyzed the Race simple main effect for each of the five age groups. To adjust for α expansion, we set the error rate of each comparison at α level = .05/5 = .01. The adults showed a significantly higher d’ for the Asain-parent  than that in the Caucasian-parent condition , t(22) = 5.165, p < .001. In children, only the oldest 11-12-year-olds exhibited a marginally significant higher d’ for the Asain-parent  than that in the Caucasian-parent condition , t(20) = 1.497, p = .074. Children in the other age groups  did not show significant differences between the two race conditions.Importantly, the Age group simple main effects (as one-way ANOVA) for both race conditions. For the Asian-parent condition, the simple main effect of Age group was highly significant, F = 13.023, p < .001, indicating an overall improvement with age for the own-race condition. The Sheffe’s method was adopted  to perform pairwise multiple comparisons and the α level was adjusted with the number of comparisons (α level = .05/10 = .005). The adults’ d’ was significantly greater than the d’s of three groups of children; adults vs. 5–6 year-olds (p < .001), adults vs. 7–8 year-olds (p < .001), adults vs. 9–10 year-olds (p < .001), and was marginally greater than the oldest children, adults vs. 11–12 year-olds (p = .028). In addition, the difference between the youngest 5–6 year-olds and the oldest 11-12-year-olds was also marginally significant (p = .008). For the Caucasian-parent condition, the simple main effect of Age group was significant, F = 3.502, p = .01. However, the posthoc pairwise comparisons revealed that only the difference between the adults and the youngest 5–6 year-olds was marginally significant (p = .009).To reveal whether a developmental change occurs—the performance improves with age for the Asian- and Caucasian-parent conditions, we also analyzed d’ showed that an own-race advantage is present in the adults and the oldest children, but is still absent in children aged between 5 and 10. Moreover, for the Asian-parent condition, we observed an overall improvement in d’ sensitivity from 5-year-olds to 11-year-olds and young adults. However, the increase in d’ sensitivity was not as apparent in the Caucasian-parent condition.In sum, the above analysis with the hits”in the SDT analyses, it is of great interest to know how well do children and adults perform at each morph level in the Asian- and Caucasian-parent conditions. Thus, we treated the morph level as a categorical variable. Our preliminary analyses revealed no significant difference between the performances of male and female participants for all age groups; we thus dropped the between-subject factor of Gender and conducted an omnibus three-way mixed ANOVA on the physically different trials only. The between-subject factor was Age Group , while the within-subject factors were Race (Asian-parent vs. Caucasian-parent) and Morph Level . The dependent measure was the rejection rate (or P(diff)), defined as the probability that the participant judged the comparison face, of a given morph level, to be “different.” The group mean rejection rates at each morph level for both race conditions are shown in the left side of As there were four morph levels in the physically “different” trials  that were collapsed as “Age group main effect was significant F = 3.49, p = .01, η2 p = .104; the group mean rejection rates were .569 (SE = .024), .592 (SE = .023), .636 (SE = .022), .680 (SE = .027), and .661 (SE = .025) for the 5–6 yr olds, 7–8 yr olds, 9–10 yr olds, 11–12 yr olds, and young adults, respectively, showing a tendency of increasing rejection rate with age. The Race main effect was highly significant F = 12.910, p < .001, η2 p = .097; the mean rejection rate was greater for the Asian-parent condition  than that for the Caucasian-parent condition . This indicated that collapsed across the age groups and the morph levels, the probability of judging the comparison face to be different was higher in the Asian-parent condition. Lastly, the main effect of Morph level was significant, F = 468.76, p< .001, η2 p = .796, the rejection rate increased as the morph level of the comparison face increased; the mean rejection rates for the morph levels 15%, 30%, 45%, and 60% were .283 (SE = .015), .569 (SE = .016), .782 (SE = .016), and .877 (SE = .014) in order.The Age Group and Race would suggest a developmental change in the own-race advantage. Indeed, the Age group*Race interaction was significant F = 3.135, p = .017, η2 p = .095, meaning that the difference in the rejection rates for Asian vs. Caucasian condition varies with age. To reveal whether certain age group exhibited an own-race encoding advantage—a higher mean rejection rate for the Asian-parent than that for the Caucasian-parent condition, we further analyzed the Race simple main effect (as paired-t-tests) for each of the five age groups, similar to the analyses with d’s stated above (the error rate was adjusted to α level = .05/5 = .01 as well). The adults showed a significantly higher rejection rate for the Asain-parent  than that for the Caucasian-parent condition , t(22) = 4.569, p < .001. M = .712, SE = .025) than that for the Caucasian-parent condition , t(20) = 1.889, p = .074. Children in the other age groups  did not show significant differences between the two race conditions. One important goal of the present study was to reveal whether certain age groups exhibited an own-race advantage. Therefore, a significant interaction between Age group simple main effects (as one-way ANOVA) for both race conditions to reveal whether a developmental change occurs. For the Asian-parent condition, the simple main effect of Age group was highly significant, F = 7.224, p < .001. Again, the Sheffe’s method was adopted to perform pairwise multiple comparisons, and the α level was adjusted to α level = .05/10 = .005. The youngest 5–6 year-olds’ mean rejection rate was marginally smaller than that of 9–10 year-olds (p = .049) and was significantly smaller than those of 11–12 year-olds (p = .002) and adults (p < .001). The 7–8 year-olds’ mean rejection rate was  smaller than 11–12 year-olds (p = .068), and significantly smaller than that of the adults (p = .008). The mean rejection rates of 9–10 year-olds, 11-12-year-olds, and the adults were not different from one another. Importantly, for the Caucasian-parent condition, the simple main effect of Age group was not significant, F = 0.878, p = .479.Likewise, we also analyzed the Age group *Morph level interaction was significant, F = 3.278, p < .001, η2 p = .099. As the morph level increased, the rejection rate increased more rapidly in adults and older children than in younger children. The group mean rejection rates for the morph levels 15%, 30%, 45%, and 60% were .293 (SE = .032), .529 (SE = .035), .696 (SE = .034), and .759 (SE = .030) for the 5–6 years olds; .289 (SE = .032), .473 (SE = .034), .732 (SE = .033), and .875 (SE = .030) or the 7–8 years olds; .312 (SE = .031), .579 (SE = .033), .781 (SE = .032), and .871 (SE = .029) in 9–10 years olds; .289 (SE = .037), .628 (SE = .040), .854 (SE = .039), and .948 (SE = .035) for the 11–12 years olds; and .233 (SE = .034), .634 (SE = .037), .846 (SE = .036), and .933 (SE = .032) for the adults, respectively. Lastly, the Race *Morph level interaction was also significant, F = 7.017, p< .001, η2 p = .055. As the morph level increased, the rejection rates increased more rapidly in the Asian-parent condition than that in the Caucasian-parent condition. The group mean rejection rates across age groups at the morph levels 15%, 30%, 45%, and 60% were .279 (SE = .018), .622 (SE = .021), .816 (SE = .017), .897 (SE = .015) in the Asian-parent condition, and .287 (SE = .019), .516 (SE = .019), .747 (SE = .021), .857 (SE = .017) in the Caucasian-parent condition. The three-way Age group *Race*Morph level interaction was not significant.The d’ stated earlier, the analyses of Age group*Race interaction revealed the presence of an own-race advantage in the adults and the oldest children, but not in the younger children under age 10. For the Asian-parent condition, we observed an overall increase of the mean rejection rate from age 5 to age 10, and the oldest children’s mean rejection rate became adult-like. On the contrary, there was no significant developmental change in the mean rejection rate for the Caucasian-parent condition. Moreover, the other two interaction effects revealed that as the morph level increased, the rejection rate increased more rapidly in the Asian-parent than that in the Caucasian-parent condition, and more rapidly for adults and older children than for younger children.In sum, similar to the findings with Morph Level of the face stimuli as a continuous variable. The performance dimension was expressed as the probability of judging the face to be “different,” while the measurements were based on the number of discrete trials at the five different stimulus intensity, 0%, 15%, 30%, 45%, and 60%. We used Sigmaplot 13.0 to conduct global curve-fitting of the group-averaged psychometric functions for each age group. Data were fit by a normal cumulative distribution function  for the Asian-parent and Caucasian-parent condition separately, with continuous points predicted by the formula below, to estimate 1) the discrimination threshold (μ), defined as the corresponding morph level yielding a rejection rate of 0.5, and 2) the slope parameter (σ).In the last part of data analyses, we treated the μ) and the slope parameters (σ) for each age group. The numbers in the parenthesis are standard errors (SE) of the estimates by bootstrap resampling with an iteration size of 200 [e of 200 . Eleven μ) was 21.58% in the Asian-parent condition and 29.72% in the Caucasian-parent condition. To reveal whether the difference was statistically significant, we used 95% confidence interval estimation (μAsian ± 1.96*SEAsian) basing on the threshold of the Asian condition (μAsian) and asked whether the discrimination threshold of the Caucasian condition (μcaucasian) fell out of the confidence interval [μAsian was 19.40%, the upper limit was 23.76%, and the threshold of the Caucasian condition μcaucasian was greater than the upper limit (29.72%>23.76%). Hence, the difference was significant. Likewise, for the slope parameter (σ)—a measure that was in inverse proportion with the steepness of the function, we used 95% confidence interval estimation basing on the Asian condition (σAsian = 13.00 ± 1.96*SEAsian) to see whether the two slopes were different. Indeed, the slope parameter in the Caucasian condition (σCaucasian = 22.33) was far above the upper limit of the confidence interval . In sum, the above analyses showed that, Taiwanese adults not only needed a smaller physical difference (in the unit of morph level) to produce a detectable change but their group psychometric function also exhibited a steeper slope  for the own-race condition.As shown in interval . The lowμ) for the Asian- and the Caucasian-parent conditions were 22.63% and 26.03%. To reveal whether the 4% difference in morph level was statistically significant, we used 95% confidence interval estimation basing on the threshold of the Asian condition (μAsian ± 1.96*SEAsian). The lower limit was 17.58%, the upper limit was 26.88%, and the threshold of the Caucasian condition μcaucasian was just below the upper limit . Nevertheless, the slope parameters (σ) of the Asian condition (σAsian = 19.37) is approaching the adult’s level, whereas the slope parameter of the Caucasian condition remained close to other children’s (σCaucasian = 27.06). The 95% confidence interval estimation revealed that the σCaucasian = 27.06 was greater than the upper limit of the confidence interval , suggesting a steeper slope of the psychometric function for the own-race condition.For the oldest 11-12-year-old group, the discrimination thresholds (μ) for the Asian- and the Caucasian-parent conditions, 28.85% and 29.77%. The slope parameters (σ) of both conditions were similar and relatively shallow, 36.87 and 39.74, respectively. The 7–8 years olds’ discrimination thresholds (μ) for the Asian- and the Caucasian-parent conditions were 29.24% and 29.87%, and their slope parameters (σ) were 28.86 and 30.17, respectively. The 9–10 years old children’s discrimination thresholds (μ) for the Asian- and the Caucasian-parent conditions were 24.29% and 26.52%, and the slope parameters (σ) were 26.99 and 28.95, respectively.For children under age 10, the 5–6 years old exhibited a similar discrimination threshold  if the discrimination threshold decreases with age, and (2) if the slope of the group psychometric function becomes steeper with age. To better present the developmental changes graphically, we put together the adults’ and children’s psychometric functions in one figure. The above descriptions were further endorsed by the 95% confidence interval estimation based on the adult’s threshold of the Asian condition. We asked whether the Children’s thresholds of the Asian condition fell out of the adult’s confidence interval, and found that all but the 11-12-year-old children’s discrimination thresholds were greater than the adult’s upper limit (>23.76%). Moreover, the 95% confidence interval estimation of the adult’s slope parameter showed that all children’s slope parameters were greater than the adult’s upper limit (>16.06). Furthermore, to reveal whether 11-12-year-old’s discrimination threshold was significantly smaller than those of other children, we used 95% confidence interval estimation and found that the thresholds of 5–6 year-olds, 7–8 year-olds, but not 9-10-year-olds were greater than the upper limit (> 26.88%). Similarly for the 95% confidence interval estimation of the slope parameter, all other children’s slope parameters exceeded the upper limit (>26.23) of the 11-12-year-old’s slope paramter.In short, the curve fitting analyses revealed that, while children aged between 5 and 10 performed equally well in both race conditions, the oldest 11-12-year-old children and adults showed better performance in the Asian-parent condition instead, indicating the presence of an own-race advantage in the latter two groups. The improvement in the perceptual sensitivity for the Asian-like morphed faces manifested itself in both lowering the discrimination threshold as well as sharpening the slope of the psychometric function, with the adults exhibiting the smallest threshold and the sharpest slope. On the other hand, the improvement in the Caucasian-like morphed faces was only observed in the slope parameter; the leftward shift in threshold was absent. Nevertheless, among all age groups, the adults still exhibited relatively better performance with the sharpest slope.d’, a smaller discrimination threshold, and a sharper slop in the Asian-parent condition than those in the Caucasian-parent condition, indicating the presence of an own-race encoding advantage. This finding well agrees with the studies by Walker & Tanaka’s and Chen et al. [Using a sequential morphing face matching task with well-controlled physical similarities between the target and the comparison faces, the present study examined the encoding advantage hypothesis in Taiwanese school-aged children and young adults. Combining the signal detection theory analysis and psychometric function curve fitting, we characterized the perceptual sensitivity for own- versus other-race faces for each age group and obtained several important findings. First, the adults showed a significantly greater n et al. ,12, and n et al.  eye moved’) and discrimination thresholds for the Asian-parent and the Caucasian-parent conditions, indicating an absence of the own-race advantage for this age range. This is also revealed in Secondly, children aged between 5 and 10 showed about equal sensitivity index  smaller threshold and a significantly sharper slope for the Asian-parent condition than those in the Caucasian-parent condition, indicating the presence of the own-race advantage. Not only they exhibited a superior performance in the Asian-parent condition, but also their psychometric functions began to look adult-like. Nevertheless, the oldest children’s proficiency in discriminating Asian-like morph faces (in terms of slope) has not yet fully reach the adult’s level.Last but not least, when comparing among all age groups, we found two distinctive developmental trends: In the Asian-parent condition, children from age 5 to 12 showed a gradual and continuous refinement in the perceptual discriminability, and the adults exhibited the best performance. In the Caucasian-parent condition, on the other hand, very limited progress was observed, and the discrimination threshold did not improve much when even reaching adulthood . Broadly speaking, these findings are consistent with the results of Chance et al. and Goodman et al., both used a standard recognition memory paradigm, 33. ChaOne main finding that the emergence of the ORE, manifested as an encoding advantage for the own-race parent condition with the oval-cropped morphing faces, did not occur before 10 or 11 years of age seem to diverge from the results reported by Pezdek et al., Sangrigoli & de Schonen, and Suhrke et al. , 16, 28,perceptual expertise hypothesis  results in people developing better expertise in distinguishing between faces of their own familiar race than those of other unfamilar races.Although face perception starts from birth, lines of evidence across decades suggested that children’s proficiency in processing faces has not yet reached the adults’ level. Carey and Diamond showed that accuracy in a match-to-sample task improves by about 20% from 6 to 10 years of age . Germine Brigham ) or the ing view  (also seing view  for a reWhat might be the reason that the ORE did not emerge until 10 or 11 years of age? Our best guess is that it seems to coincide with the age of children reported to exhibit an adult-like ability in face processing. One characteristic of the adult-like expertise in face recognition is the priority of using internal facial cues as opposed to using external cues. For example, Campbell et al. asked 4- to 10-year-old children to make familiarity judgment of part-face and whole-face of their schoolmates and found that external features were more accurate than internal features for younger children, with a switch to an adult-like pattern at age 9 or 10 . In the Another important feature of an adult-like face processing is the mastering of configural processing . In the perceptual expertise or perceptual learning hypothesis of ORE. Important avenues for future research are to extend the age range to adolescence, to increase sample size and regard age as a continuous variable, to obtain convergent evidence of participants from a different race/ethnicity, and to explore how the visual own-race bias may link to the development of race-based social preference/prejudice of “in-group” vs. “out-group” distinction [In conclusion, the present study is not the first to adopt the morph face paradigm in studying the other-race effect, but perhaps is the first to examine school-age children’s perceptual sensitivity for own- and other-race morph faces by estimating the discrimination thresholds and slope parameters with psychophysical methods. Our results support the view that expertise in face processing takes the entire childhood to fully develop, and the emergence of the own-race encoding advantage coincides well with the onset of an adult-like expertise in processing face identity. Lastly, the other-race effect seen in adulthood may reflect a result of prolonged learning specific to own-race faces most commonly seen in one’s visual environment—a finding supporting the tinction , 51.


To determine whether high signal-to-noise ratio (SNR) acquisitions improve the repeatability of liver proton density fat fraction (PDFF) measurements using confounder-corrected chemical shift-encoded magnetic resonance (MR) imaging (CSE-MRI).F-test and t-test, respectively. Two reconstruction algorithms (complex-based and magnitude-based) were used for both the phantom and in vivo experiments.Eleven fat-water phantoms were scanned with 8 different protocols with varying SNR. After repositioning the phantoms, the same scans were repeated to evaluate the test-retest repeatability. Next, an in vivo study was performed with 20 volunteers and 28 patients scheduled for liver magnetic resonance imaging (MRI). Two CSE-MRI protocols with standard- and high-SNR were repeated to assess test-retest repeatability. MR spectroscopy (MRS)-based PDFF was acquired as a standard of reference. The standard deviation (SD) of the difference (Δ) of PDFF measured in the two repeated scans was defined to ascertain repeatability. The correlation between PDFF of CSE-MRI and MRS was calculated to assess accuracy. The SD of Δ and correlation coefficients of the two protocols (standard- and high-SNR) were compared using in vivo study demonstrated that the repeatability of the high-SNR protocol (SD of Δ = 0.53 for complex- and = 0.85 for magnitude-based fit) was significantly higher than using the standard-SNR protocol . No significant difference was observed in the accuracy between standard- and high-SNR protocols.The phantom study demonstrated that higher SNR improved the repeatability for both complex- and magnitude-based reconstruction. Similarly, the Higher SNR improves the repeatability of fat quantification using confounder-corrected CSE-MRI. Similar to alcoholic fatty liver disease, nonalcoholic fatty liver disease (NAFLD) can progress to inflammation and fibrosis, eventually resulting in cirrhosis.4 However, biopsy is invasive, expensive, and unsuitable for longitudinal treatment monitoring.6 Further, biopsy suffers from high variability for quantitative assessment of liver disease, including steatosis.8 Hence, alternative methods have been proposed for liver fat quantification, including ultrasound-9 and computed tomography-based methods,11 and magnetic resonance imaging (MRI)-based chemical shift-encoded (CSE) methods.14An accurate and precise (i.e. repeatable) method to detect and monitor hepatic steatosis is urgently needed for the management of patients with NAFLD. Non-targeted percutaneous liver biopsy is the current reference standard to detect hepatic steatosis and definitely diagnose NAFLD.15 Combined with other imaging techniques including hepatobiliary contrast agents for assessment of focal liver lesions17 and magnetic resonance (MR) elastography for assessment of liver fibrosis,18 liver fat quantification using CSE-MRI19 contributes to the comprehensive assessment of liver disease.20 By addressing all relevant confounding factors,24 CSE-MRI provides quantitative maps of proton-density fat-fraction (PDFF), a well-validated biomarker of triglyceride concentration, over the entire liver.27 As has been demonstrated in multiple recent studies, CSE-MRI provides accurate and reproducible liver PDFF quantification across different vendors and field strengths,29 over a broad range of PDFF (e.g. 0–50%).Recently, MRI is increasingly used for the evaluation of the liver either for focal liver lesion and diffuse liver disease.30 a fat-fraction of 5.56% was established as the 95% threshold in a patient population with no identifiable risk factors for steatosis. Tang, et al. suggested the cutoff value of 6.5% in PDFF to discriminate steatosis grade ≥1 from grade 0.31 Another recent study identified a threshold of 4.96% for indicating substantial macrovesicular steatosis in pathological specimens.32 More recently, an even lower threshold (PDFF = 3.5%) was shown to be highly predictive of metabolic syndrome in adolescent females.26However, recent studies suggest the need for accurate and precise PDFF quantification at low fat-fractions. In the Dallas-Heart study,28 or ±1–2 pp using CSE-MRI with breath hold and respiratory-triggering methods.33 These relatively broad confidence intervals may limit the utility of CSE-MRI for fat quantification over the low PDFF range (e.g. 0–10%).Reliable PDFF quantification near these low thresholds requires highly accurate and precise techniques. Previous studies established the repeatability  of CSE-MRI as ±2–4 percent point (pp) at 1.5T and 3T,3 at 1.5T.34 High spatial resolution is required to identify anatomical or pathological objects, e.g. vessels or lesions, for accurate PDFF/35 Therefore, we hypothesize that increasing SNR through small reductions in spatial resolution may improve the test-retest repeatability of CSE-MRI without affecting its accuracy.Sufficient signal-to-noise ratio (SNR) is a key factor required for repeatable measurements. SNR has a direct relationship with voxel size. In general, the voxel size of standard CSE-MRI acquisitions is ∼10–37 mmin vivo liver imaging.Hence, the purpose of this study was to demonstrate the relationship of increased SNR and improved repeatability of CSE-MRI-based PDFF measurements. The relationship between SNR and repeatability was evaluated using theoretical analysis, fat-water phantom imaging, as well as 37 For this estimation, we assumed the following imaging parameters for CSE-MRI: 1.5T, 6 acquired echoes acquired with minimum TE of 1.23 ms with echo spacing of 1.88 ms , a multi-peak fat signal model with six peaks,38 true PDFF = 5%. In these calculations, voxel-wise echo image SNR  was varied from 5 to 300 to assess the relationship between SNR and repeatability. For these calculations, PDFF measurements were considered over an ROI consisting of 117 voxels . CRB-based calculations of repeatability (standard deviation of the difference between ROI-based measurements of PDFF) were calculated as PDFFSD is the theoretical standard deviation of voxel-wise PDFF measurements obtained at each SNR level, measured both for complex-fitting and for magnitude-fitting based PDFF quantification.Test-retest repeatability (standard deviation between repeated acquisitions) was estimated based on Cramér-Rao Bound (CRB) calculations.39 The phantom was comprised of 11 cylindrical glass vials  with oil-water emulsions including nominal fat fraction (FF) of 0–10% by 1% . Each vial contained 40 mL of oil-water emulsion, where the water component included agar (2% w/v) to form the gel, CuSO4 (3 mM) to shorten the T1 of water, sodium dodecyl sulfate (43 mM) as surfactant, NaCl (43 mM) to adjust the conductivity, and sodium benzoate (3 MM) as preservative.A fat-water phantom was constructed, consisting of multiple vials with agar based emulsions of peanut oil and water, similar to previous works.yk acquisition of 30%, bandwidth of ±167 kHz.High spatial resolution with low SNR, with voxel size of 1.6 × 1.6 × 1.5 mm, partial Intermediate spatial resolution with intermediate SNR, acquired with identical parameters except 1.9 × 1.9 × 4.0 mm voxel size and bandwidth of ±91 kHz.Low spatial resolution with high SNR, acquired with identical parameters except 2.5 × 2.5 × 6.0 mm voxel size and bandwidth of ±63 kHz.Imaging was performed on a clinical 1.5T scanner  with an 8-channel body coil. The CSE-MRI acquisitions were performed repeatedly using a three dimensional (3D)-spoiled gradient echo acquisition, with eight different protocols by changing the spatial resolutions and number of excitations (NEXs). After repositioning the phantoms, the same scans were repeated to evaluate test-retest repeatability. MR parameters were designed with three protocols with varying spatial resolution:High and medium resolution protocols were scanned with 1, 4, and 16 signal averages (NSA), and low resolution protocols with 1 and 4 NSA. Other acquisition parameters included: repetition time (TR) of 21.2 ms; maximum and minimum echo time (TE) of 10.5–10.7 ms and 1.1–1.2 ms ; 32 slices; 36 cm field of view; five degree flip angle. Parallel imaging was not used for the phantom experiments.S are the complex signal intensities within the ROI of the FF = 0% phantom and S’ is the signal from real channel of the background noise outside the vials, to avoid bias related to Rician noise distribution.41The SNR of the eight different protocols were estimated by the following equation:in vivo study was performed after obtaining approval from our local Institutional Review Board. Written informed consent was obtained from all subjects. 20 healthy subjects  were prospectively recruited. Further, 28 patients  who were scheduled for routine clinical abdominal MRI were also recruited prospectively. Exclusion criteria included any contraindication to MRI and age less than 18 years.This prospective 2-corrected STEAM spectroscopy (MRS) as the reference standard for PDFF. The first two acquisitions (a and b) were repeated after removing the subject from the scanner bore, removing the anterior coil elements, sitting the subject up on the table, allowing the subject them lie down again and replacing the coil. Every sequence was acquired during a single breath hold (19–26 s). The entire liver was imaged using a 3D volume oriented in the axial plane for both the standard and high SNR protocols. All data were reconstructed with both complex- and magnitude-based fitting as we did for phantom study.All imaging was performed on a clinical 1.5T scanner  with an 8 or 12 channel phased array coil. The following three quantitative acquisitions were performed  MR spectroscopy (MRS) acquisitions were obtained over a single 2.0 × 2.0 × 2.0 cm37 In both cases, the confounding factors of 23 accurate spectral modeling of fat22 and correction for eddy currents44 were addressed. Low flip angles (2–5 degree) were used to minimize T1 bias.21All data were reconstructed using both complex and magnitude based water-fat reconstructions to produce quantitative PDFF maps over the entire liver.2 in the center of each of the 11 phantoms.For phantom experiments, PDFF measurements were obtained by placing a circular region of interests containing ∼420 mmin vivo study, a radiologist with 13 years’ experience in liver imaging placed ROIs in the posterior segment of the right lobe of the liver. A circular ROI was placed in the liver on PDFF maps from CSE-MRI and was co-registered with the MRS voxel as closely as possible. During the ROI placement, special attention was paid to avoid partial volume effects at the liver edge, obvious artifacts or contamination of large vessels. ROI placement was manually adjusted if any obvious image artifacts such as ghosting were identified at that location.For 2 decay across spectra at different TEs.45Fat-quantification from MRS data was performed using an in-house fitting routine that accounts for the spectral complexity of the fat signal as well as for TTo evaluate the test-retest repeatability, we first defined the difference (Δ) as the difference of PDFF measured between test and retest acquisitions. Here we assumed that ideal mean of Δ would be 0 and standard deviation of Δ would estimate the variability observed in repeated measurements, i.e. lower standard deviation of the Δ represents higher precision (improved repeatability).in vivo study, the repeatability (standard deviation of Δ) of CSE-MRI with the standard SNR protocol was compared with the high SNR protocol using F-test.The results of repeatability (standard deviation of the Δ) in phantom scans were plotted in single logarithmic graph against SNR. In the t-test after Fisher’s r-z transformation.To evaluate the accuracy of PDFF measurement, linear regression analysis was performed. The correlation coefficients (r) of PDFF with CSE-MRI and MRS were calculated and compared by P-value of <0.05 was considered to be statistically significant. Statistical analyses were performed using MedCalc  and R version 3.1.1 (R Foundation Statistic Computing).The correlation coefficients were interpreted as no correlation for 0–0.20, fair correlation for 0.21–0.40, moderate correlation for 0.41–0.70, substantial for 0.71–0.90, and strong correlation for 0.91–1.0. A The results of repeatability (standard deviation of Δ) in phantom study were shown in SNR estimation from the Monte-Carlo based pseudo-multiple replica method revealed that high SNR protocol of CSE-MRI had ∼3.3 times more SNR than standard SNR protocol at scanner 1 and ∼2.7 times at scanner 2 .P < 0.001; and 0.94 pp for magnitude-based fit, P = 0.003) (As shown in = 0.003) .r = 0.985–0.987). (Correlation between the PDFF of MRS and CSE-MRI were very strong in both standard and high SNR protocols (–0.987).  and 4 No–0.987). .in vitro in phantom experiments and in vivo in human studies that increasing the SNR of a CSE-MRI acquisition by increasing voxel size improves the precision of PDFF quantification, with no impact on fat quantification accuracy. This has important implications for the use of confounder-corrected CSE-MRI methods aimed at diagnosing hepatic steatosis and for longitudinal treatment monitoring. By using acquisition protocols with higher SNR performance, more precise quantification of liver fat can be made without creating additional bias that degrades accuracy.In this work, we have demonstrated theoretically, In the phantom study, we showed that MR parameter settings that produced higher SNR provide better test-retest repeatability in PDFF measurements made with CSE-MRI. Theoretical SNR performance, as determined by CRB analysis, demonstrated close agreement with repeatability measurements made in phantoms.in vivo clinical study.High SNR protocols, which had 2.7–3.3 times more SNR than standard SNR protocol, showed significantly improved repeatability than standard SNR protocol. Importantly, the use of a high SNR protocol did not affect the accuracy (ie: lack of bias) in either the phantom or 3, avoiding parallel imaging , and reducing bandwidth, all of which improve SNR performance.Acquisitions that are both accurate and repeatable are necessary for quantitative imaging methods such as PDFF quantification in the liver. One practical solution for improving the estimation of true value is reducing noise in the image. Decreasing the spatial resolution and increasing the sampling interval (decreasing bandwidth) are simple and effective way to increase SNR in MRI. As hypothesized, PDFF measurements made using CSE-MRI became more robust by increasing the voxel size from ∼35–50 to ∼115∼124 mm28 Another study showed that between the different acquisition methods (breath-hold and respiratory-triggering) was ∼0.9 pp.33 In our study, the repeatability using standard SNR protocol (0.77–0.93 pp) was slightly better than those in previous studies, probably because the same acquisition and the same scanner were used. Using the high SNR protocol, however, repeatability of complex-fit was 0.37 pp, which means that the 95% confidence interval of Δ is less than 1 pp (−0.62 ± 0.84 pp). The high repeatability of PDFF measurements, achieved with the high SNR protocol, would enable a precise assessment of PDFF near the threshold that is required for the assessment in patients with low liver concentrations.In previous studies, the standard deviation of the difference in the PDFF measurement between 1.5T and 3T was ∼1.4 pp.in vivo study was 0.37 pp observed in a high SNR protocol using complex-based fit even though the SNR of the high SNR protocol were >78. This indicates that there must be other factors that affect repeatability such as biological factors like motion. Inconsistent breath-holding or other motion can be another factor that can impact the apparent accuracy and precision. Further, we also speculate that spatially varying fat concentration in the liver may be a major factor. Since perfect co-registration between two scans is not possible, subtle heterogeneity of the fat distribution in the liver would adversely impact the apparent variability of a PDFF measurement.In the phantom study, the theoretical repeatability-SNR curve was in very good agreement with experimental measurements. These results also demonstrate a repeatability (standard deviation of Δ) of less than 0.1 pp if SNR is >70–100. However, the best repeatability 46 The standard SNR protocol usually used parallel imaging to achieve reasonable spatial resolution. Spatially varying noise amplification from the parallel imaging reconstructions, referred to as the g-factor, prevented the use of conventional SNR estimation methods where the noise is estimated using a ROI from a background region.47 By using a Monte Carlo approach, properly scaled and correlated noise is repeatedly added to the raw data to generate a stack of replicas. Voxel-wise estimates of the noise can be made by making voxel-wise measurements standard noise through the stack of replicas.We applied a Monte Carlo approach to estimate the SNR of the in vivo scan.2-weighted image and contrast-enhanced images, for the anatomical and/or morphological assessment. However, further study is necessary to determine the appropriate parameters for CSE-MRI by assessing both quantitative role as PDFF measurement and qualitative role for anatomical evaluation. Further, the use of multiple ROI’s to estimate PDFF over the liver can be used to improve the repeatability of PDFF estimates in the. However, a detailed analysis of the impact of the size and number of ROI’s on the repeatability of PDFF estimates, while relevant, is beyond the scope of this study. Another limitation is that we could not address the effect of R2 star values in the liver. During the acquisition of CSE-PDFF, six sequential echoes were obtained to correct T2 star effect of the liver. However, if the huge amount of iron accumulates in the liver, that may affect the repeatability of CSE-PDFF. Unfortunately, there was no large variety in R2 star values (22–52/s in right lobes of the livers), which were not large enough to study the effect of T2 star effect. Further study with large amount of iron in phantom/subjects are necessary to deal with this issue.This study had several limitations. First, we did not evaluate the effect of decreased spatial resolution on clinical decision by radiologists, although the change in spatial resolution was relative small. In the liver MRI, we typically obtain other sequences, e.g. TIn conclusion, the use of higher SNR CSE-MRI acquisitions improves the precision of quantitative PDFF measurements in the liver, without negatively impacting the accuracy of these measurements.


D. radiodurans. We showed that overexpression of the toxin MazF-dr inhibited the growth of Escherichia coli. The toxic effect of MazF-dr was due to its sequence-specific endoribonuclease activity on RNAs containing a consensus 5′ACA3′, and it could be neutralized by MazE-dr. The MazF-dr showed a special cleavage preference for the nucleotide present before the ACA sequence with the order by U>A>G>C. MazEF-dr mediated the death of D. radiodurans cells under sub-lethal dose of stresses. The characteristics of programmed cell death (PCD) including membrane blebbing, loss of membrane integrity and cytoplasm condensation occurred in a fraction of the wild-type population at sub-lethal concentration of the DNA damaging agent mitomycin C (MMC); however, a MazEF-dr mutation relieved the cell death, suggesting that MazEF-dr mediated cell death through its endoribonuclease activity in response to DNA damage stress. The MazEF-dr-mediated cell death of a fraction of the population might serve as a survival strategy for the remaining population of D. radiodurans under DNA damage stress.Here we identified a functional MazEF-dr system in the exceptionally stress-resistant bacterium  Toxin–antitoxin (TA) systems, also referred to as addiction or suicide modules, are distributed widely in free-living organisms including bacteria and archaea . TA systEscherichia coli . The viable fractions of D. radiodurans in response to stresses exhibited a characteristic sigmoid dose-response curve with a “shoulder” representing resistance at low doses and specially a “steep slope” representing substantial decrease of viability at sub-lethal and higher doses. The sigmoid dose-response curve distinguishes the extremophiles from damage-sensitive bacteria such as E. coli with the exponential dose-response curve , protect essential proteins such as DNA repair enzymes from oxidative damage. Second, efficient DNA repair mechanisms are required for the recovery of a fully functional genome, for instance, RecA and PPrI (IrrE) are critical repair proteins in homologous recombination pathway of D. radiodurans. Moreover, a combined mechanism including efficient DNA, protein, membrane lipid protection and repair was proposed to be required for the organism’s response to extreme radiation  as query sequences. Two pairs of putative MazEF-encoding genes were found in the D. radiodurans genome: dr0416-dr0417 and dr0661-dr0662, which are consistent with the predicted sequences in the Toxin-Antitoxin Database (TADB)  . DR0417 E. coli, and the inhibition of cell growth was monitored. Western blot assays with anti-DR0417 and anti-DR0662 antibodies confirmed that the proteins were expressed successfully in E. coli . In contrast, cells with co-overexpression of DR0416 and DR0417 or those with over-expression of DR0416 alone showed similar growth profiles to that of the control, indicating that the DR0417-induced growth defect could be neutralized by DR0416 . However, over-expression of DR0662 and/or DR0061 in E. coli did not result in obvious changes of cell growth . These results indicated that the dr0416-dr0417 locus may function as MazEF-type TA system and we therefore refer to them as MazEF-dr for MazEF in D. radiodurans.To determine whether two pairs of MazEF homologs are functional TA systems, each MazEF homology module was overexpressed in in vitro activity of MazF-dr was assessed. Total cellular RNAs were extracted from D. radiodurans cells. Following incubation with purified MazF-dr, a gradual accumulation of cleavage products appeared, and they increased with increasing protein concentration . Especially 16S rRNA and 23S rRNA of D. radiodurans were cleaved completely into smaller fragments by MazF-dr at high concentration , while few cleavage products of 5S rRNA and tRNAs were detected. When an exogenous RNA  was used as a substrate of MazF-dr, a specific cleavage profile was also observed . Thus, MazF-dr seems to be an endoribonuclease that cleaves RNAs. However, pre-incubation of MazF-dr with MazE-dr completely inhibited the RNase activity of MazF-dr , indicating that the activity of MazF-dr could be neutralized by MazE-dr. These results suggest that MazF-dr acts as the toxin and MazE-dr is the antitoxin that might abrogate the toxic effect of MazF-dr.To evaluate the biochemical properties of MazEF-dr, the Figures D, two cleavage patterns were identified, both of which consisted of an ACA consensus sequence . In the first pattern , cleavage occurred at the 5′ end of an adjacent A in the tetrad sequence (↓A↓ACA). For the other cleavage pattern , cleavage occurred at the 5′ end of the A (↓ACA). Notably, the cleavage activity of MazF-dr at the A↓ACA site was higher than that at the ↓AACA site, as determined by the product intensity, suggesting that the ↓ACA is the major cleavage site.A primer extension assay was used to analyze the RNA cleavage site of MazF-dr. 16S rRNA was used as the substrate to determine the cleavage site of MazF-dr. As shown in Figure 4A, MazF no longer cleaved the RNAs that lacked the ACA consensus sequence. These results indicated that MazF-dr specifically cleaved RNAs containing a consensus 5′ACA3′ sequence in a ribosome-independent fashion. In addition, substitutions of the nucleotide before the ACA sequence with A, G, U, or C did not prevent cleavage function of MazF-dr, but it affected the cleavage activity of MazF-dr . MazF-dr had the highest cleavage activity on the U-substituted sequence, showing a decreasing cleavage activity in the order of U>A>G>C, suggesting that the surrounding nucleotide composition of the substrates has the impact on the activity of the toxin. Unlike D. radiodurans MazF, EcMazF did not have a cleavage preference for the nucleotide before the ACA consensus sequence .To further validate the consensus cleavage sequence and the effects of the 5′ terminal nucleotide adjacent to the ACA sequence of substrates on the activity of MazF-dr, we synthesized a series of 5′ fluorescein amidite (FAM)-labeled RNA derivatives . Under low doses of DNA damaging agents , the dose-response curve of the wild type and ΔMazEF-dr showed a similar “shoulder,” representing little decrease in the viability; however, a “steep slope” representing a substantial decrease of the viable fraction of the wild-type strain was observed at higher doses of the DNA damage agents , compared with ΔMazEF-dr cells . Mutants of a DNA repair related gene (pprI) and an antioxidant related gene (crtB), which was involved in the carotenoid biosynthesis, were used for the comparison purpose . The pprI mutant was more sensitive to MMC stress than those of the wild type and ΔMazEF-dr, while the crtB mutant was as resistant as the wild type.We evaluated the roles of the Figure 6A). The living cells were stained by SYTO 9 (green) and the dead cells were stained by propidium iodide (PI) (red). Treatment with a sub-lethal dose of MMC (15 μg/ml) led to the death of a substantial number of the wild-type cells, while most of the mutant cells remained alive after the treatment. A transmission electron microscopy (TEM) analysis demonstrated that morphological changes occurred in 24%  of the wild-type cells, compared with the mutant cells under sub-lethal dose of the MMC stress , including the loss of membrane integrity, membrane blebbing and cytoplasm condensation, which are the characteristics of PCD , suggesting that the MazEF-dr mediated cell death of a fraction of the population in response to sub-lethal dose of DNA damage stress.The cell viability data are consistent with the cell phenotypes obtained by a confocal microscopy assessment using the Live/Dead Kit   , and Mazectively , while M↓ACUCA3′ . The conrculosis . In our E. coli MazF cleaved RNAs without an obvious preference for the nucleotide preceding the consensus ACA sequence. These results indicate that MazF-dr has a special substrate-preference that depends on the surrounding nucleotide composition of the substrates. The roles of the substrate-preference of MazF played in response to DNA damage stress are still unclear.Substitution of the 5′ terminal nucleotide before the ACA sequence with A, G, U, or C did not prevent the cleavage function of MazF-dr, but it did affect the cleavage activity of MazF-dr, which showed the highest cleavage activity on the RNA with the U substitution. In contrast, the mazEF modules are involved in stress-response of some pathogens, but the mechanism by which bacteria deal with stresses is still unclear  above sub-lethal doses, which are characteristics of the sigmoid dose-response curve of extremophiles. The sigmoid dose-response curve and distinguishes the extremophiles from damage-sensitive bacteria with the exponential dose-response curve , suggesting that MazEF-dr mediates the suicide of a subpopulation and the survival of the remaining population under the stress. MazEF-dr-mediated cell death of a fraction of the population provides an explanation for the canonical sigmoid dose-response curve. The expression levels of DNA repair-related genes, including recA and ddrO, did not changed significantly under the sublethal dose of MMC  as the quorum-sensing signals employed by D. radiodurans, which are induced upon cell exposure to high levels of oxidative stress  pLysS were used for cloning and protein expression studies, respectively. E. coli BW25113Δ6 (kindly provided by Prof. Nancy A. Woychik) was used for cell growth inhibition experiments. E. coli strains were cultured in Luria-Bertani (LB) broth or LB agar at 37°C unless otherwise noted. The media were supplemented with chloramphenicol  or kanamycin  if necessary. D. radiodurans and its derivatives were cultured in TGY  broth or TGY agar at 30°C. Streptomycin  was added to the cultures of D. radiodurans mutant strains.The bacterial strains and plasmids used in this study are listed in Supplementary Table BamHI and HindIII, respectively, were PCR-amplified with the corresponding primers listed in Supplementary Table 2 method. Mutants were screened on TGY plates containing streptomycin, and then all the recombinants were confirmed by PCR analysis and sequencing.Mutant was constructed by double crossover recombination of a streptomycin resistance cassette into the genome as described previously . BrieflyE. coli BW25113Δ6 cells, and their expression was induced by arabinose and isopropyl-β-D-thiogalactoside (IPTG), respectively. Cultures were grown to an optical density at 600 nm (OD600) of 0.6, then 0.2% arabinose and/or 0.3 mM IPTG were added to induce the expression of the toxins and/or antitoxins. E. coli cells transformed with pBAD33-dr0417 and pET28a-dr0416, or pBAD33-dr0662 and pET28a-dr0661 were cultured overnight in LB broth. Then, the cultures were diluted into fresh LB broth and grown to OD600 of 0.55. Subsequently, each culture was divided into four equal parts. One of the cultures served as control, to which was added equal ddH2O, while 0.2% arabinose, 0.3 mM IPTG or both 0.2% arabinose and 0.3 mM IPTG were added to the other three cultures to induce the expression of toxin and/or antitoxin. These cultures were incubated at 37°C and the OD600 was measured hourly to evaluate the cell growth.The toxin and antitoxin homology genes were cloned into pBAD33 and pET28a, the resulting plasmids were transformed or co-transformed into D. radiodurans exposed to DNA damage stresses was determined as described previously . For the MMC  treatment, the cells were treated with 5–20 μg/ml MMC, and samples were taken after 40 min and plated on TGY agar plates after being diluted appropriately. For the H2O2 treatment, the cells were treated with different concentrations of H2O2 for 30 min. After incubation, the reaction was stopped by catalase (20 U) for 15 min. Then, the cells were diluted and plated on TGY agar plates. For the γ-radiation treatment, cell suspensions were irradiated at room temperature for 2 h with 60Co γ-ray  at several different doses (from 3 to 12 kGy), which were adjusted by changing the distance of samples from the γ-ray source  glycerol] containing PMSF  and lysed by sonication. Cell lysates were centrifuged at 15,000 × g for 30 min to remove debris, and the supernatant was loaded onto a Ni-NTA column , the target proteins were eluted with elution buffer B . The collected protein fractions were dialyzed against buffer C  and then loaded onto HiTrap Q ion exchange column  and eluted by gradient elution. Finally, proteins were further purified on a Superdex 75 column  with buffer C. Fractions containing the target proteins were pooled, concentrated, flash-frozen in liquid nitrogen, and stored in -80°C.DR0416 and DR0417 were expressed from a pET28a+ vector containing a N-terminal 6×His tag in BL21 (λDE3) pLysS cells. When cells grown in LB media reached an ODD. radiodurans was isolated using the TRIzol method according to the manufacturer’s protocol . A culture of D. radiodurans was grown in TGY medium until the OD600 reached ∼0.6. Then, the cells were pelleted and resuspended in 1 ml of TRIzol solution. After vortexing, the samples were incubated for 15 min at room temperature.Total RNA of D. radiodurans total RNA or MS2 phage RNA (RefSeq accession no. NC_001417) at 37°C for 15 min. The reaction mixture consisted of DR0417, RNA, 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 μl of RNase inhibitor  in 20 mM Tris-HCl (pH 8.0), with or without DR0416. After denaturation in urea, the mixtures were loaded onto a 4.5% polyacrylamide gel containing 8 M urea, electrophoresed, and then stained with ethidium bromide (EB).Purified DR0417 was incubated with Deinococcus radiodurans 16S rRNA was transcribed in vitro using the T7 promoter method . Then DNase I (New England Biolabs) was used to digest the 16S rDNA substrate. The obtained 16S rRNA products were extracted using phenol-chloroform and precipitated with ethanol.r method . Brieflyr method . After p2 . Primer extension was carried out in 10 μl of the reaction mixture containing 5′-FAM labeled primers . The endoribonuclease activity of MazF was assayed in a 10 μl reaction mixture containing MazF, FAM-labeled RNA substrates, 0.2 μl of RNase inhibitor , 20 mM Tris-HCl (pH 8.0), and 0.1 mM EDTA. The reaction mixture was incubated at 37°C for 15 min, and stopped by adding 2 μl of RNA loading buffer . Then, the reaction mixture was loaded onto a 20% urea PAGE and scanned by the Typhon system .600 of 0.8 were washed twice with 0.85% NaCl. Cells treated with a sub-lethal dose of MMC , as well as untreated cells, were collected by centrifugation. Then the cells were stained using 1 μl of a 1:1 mixture of PI and Syto 9 from the kit and were incubated for 15 min at room temperature keeping in the dark. Following the wash by 0.85% NaCl twice, 100 μl of 4% formalin was applied for the fixation. The cells were washed twice with PBS buffer and re-suspended in 20 μl of 50% glycerol. A confocal microscopy assay was performed using a Leica DM4000 fluorescence microscope. Living cells were stained by Syto 9 (green) and death cells were stained by PI (red).The cell death was assessed by using confocal microscopy. The cells were stained using the Live/Dead Kit containing PI and SYTO 9 (Invitrogen). Cells grown to an ODD. radiodurans cells treated with MMC , as well as untreated cells, were collected by centrifugation. Samples were processed as described previously (For the TEM analysis, eviously . The celTL, BT, and YH were responsible for the experiments design and drafted the manuscript. XM revised the manuscript finally. SD and YJ constructed the vectors and mutants. The viability assays were completed by ML. JL and JY performed the primer extension assays. TL performed the confocal microscopy and TEM observation and all data analysis. All authors reviewed the manuscript and approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vagal nerve stimulation (VNS) for refractory epilepsy is well established. Trigeminal neuralgia itself is a common disease in adults, and thus, late-onset pain in the trigeminal region under VNS, which is extremely rare, may not be recognized as caused by VNS.Two patients with drug-resistant symptomatic epilepsy treated with chronic VNS experienced stimulation-related pain in the lower and upper jaw and teeth on the side of stimulation. No evidence of local spread of the stimulation current was present. The pain started with a delay of years after device implantation and weeks after the last increase in the pacing parameters. At the time of onset, the pain was not recognized as VNS-related, leading to extensive examinations. The trigeminal neuralgia-like pain resolved after adjustment of the stimulation current intensity. In one of the patients, the pain disappeared within one to two days following every epileptic seizure. To our knowledge, this is the first case report of late-onset trigeminal pain under VNS revealing a direct link between epileptogenic and pain processes.A painless interval between the last change of the pacing parameters and trigeminal pain can lead to the erroneous interpretation that this is a typical trigeminal neuralgia. The lack of its recognition as a side effect of VNS can lead to unnecessary examinations and delayed adjustment of stimulation parameters. In patients with signs of late-onset trigeminal pain under VNS with normal electrode impedance and no evidence of local current spread, the replacement of the VNS lead does not seem to be beneficial. A review of the literature on VNS side effects including pain and device malfunctions was undertaken. Vagal nerve stimulation (VNS), delivered by the NCP System  for treatment of drug-resistant epilepsy is approved as an add-on therapy in adults and children for partial and generalized epileptic seizures. New, noninvasive stimulation devices are under development , 2. The The adverse events (AE) of VNS are of two types: implantation procedure-related and stimulation-related. Surgery-related AE have been reported in 3-22% of VNS implantations. The most often reported surgery-related AE are hardware failure in 3.7-16.8%, lead fracture or disconnection in 3.7-13.7%, wound infections in 1.7-7.1%, wound hematoma in 0.7-1.9%, transient asystole/bradycardia up to 1%, left vocal cord palsy, mostly transient, in 1.4-5.1%, and lower facial weakness in 0.2-1.2% . StimulaAt the University Hospital Bratislava, Slovakia, we implanted VNS systems in 54 patients with drug-resistant epilepsy not amenable to resection epilepsy surgery between April 2009 and December 2016. Forty-seven of the patients were followed for a long time (one to eight years). The VNS systems were implanted on the left side, and patients had regular follow-up visits with a stepwise increase in stimulation current by 0.25 mA in 4-8 weeks. The target range of the stimulation current intensity, if tolerated, was between 1.25 and 2.00 mA with a stimulation frequency 25-30 Hz, pulse width of 250-500 μsec and a duty cycle of 30-21 s on and 5 to 1.1 min off, as recommended by the manufacturer.Two of our patients perceived stimulation-related pain in the upper and lower jaw and teeth ipsilateral to the side of stimulation with a delay of years after device implantation and weeks after last augmentation of the stimulation current intensity, thus mimicking coincidental trigeminal neuralgia to the VNS Table . Both weA 46-year-old man with intractable symptomatic bitemporal epilepsy lasting 33 years, with MRI-verified right temporooccipital periventricular heterotopy, was implanted with VNS in November 2012. His seizure frequency before implantation was up to 10 motor seizures with impaired awareness per month and sporadic bilateral tonic-clonic seizures. After implantation, the stimulation current was increased stepwise in 4-8 weeks. He achieved a 50% reduction in seizures with 2.0-mA stimulation current, 30-Hz frequency, 500-μsec pulse width, and 30-s on-time and 1.8-min off-time. Eighteen months after initiation of VNS stimulation, two months since the last increase in the stimulation current, the patient began to complain of sharp, shooting pain in the upper and lower jaw and teeth on the left side, without trigger points or a sensory deficit. The patient underwent CT scans and detailed dental examination, but no pathological processes were discovered. It was the patient who first noticed that the painful shootings were regular, lasting for tens of seconds with the stimulation on, and that the painless intervals lasted minutes with the stimulation off. The consequent analysis, when we checked the painful and pain-free intervals, ascertained that the pain was associated with the stimulation period of VNS. When the device was off, he was pain-free. The impedance of the electrode was normal. The intensity of the pain was evaluated as 10 on a visual analogous 10-point scale (VAS). The patient had no signs of local current spread to the surrounding tissues . The pain mimicked trigeminal neuralgia type 1 according to Burchiel’s classification . AccordiA 50-year-old woman with intractable symptomatic bitemporal epilepsy lasting 37 years, with MRI-verified bilateral cystic malformations of mesial temporal lobe structures, was implanted with VNS in April 2011. Her seizure frequency was up to 6 motor seizures with impaired awareness per month and sporadic bilateral tonic-clonic seizures. After VNS implantation, the stimulation current was increased stepwise in 4-8 week intervals up to a 1.0-mA stimulation current intensity, 30-Hz frequency, 250-μsec pulse width, 30-s on-time and 3-min off-time. At these stimulation parameters, she was a 50% responder. Four years later, she overcame status epilepticus. The stimulation current was increased to 1.25 mA and the duty cycle to 30 s on and 1.8 min off. Two weeks later, she began to complain of shooting, sharp pain in the upper and lower jaw and teeth on the left side, without a trigger point or sensory deficit. The intensity of the pain was evaluated as an 8 on the VAS. The pain mimicked trigeminal neuralgia type 1 according to Burchiel’s classification . The patCarius and Schulze-Bonhage  reportedIn our patients with late-onset jaw and dental pain, the gradual increase in the stimulation current intensity up to 1 mA and more was uncomplicated. The pain appeared after a pain-free interval from the last adjustment without any accident or trauma and no signs of local current spread. After a 6-month stimulation-free period, patient 1 regained the tolerance for stimulating current up to 1.5 mA, thus pointing to possible desensitization. A unique, intermittent course of stimulation related pain was documented in him with pain relief for 24-48 h following every epileptic seizure. Mechanisms of activity-dependent central sensitization are likely explanations . The nucWith reference to the data accumulated in previous years, late-onset trigeminal pain under VNS stimulation in our patients and patients reported previously can be explained by mechanisms of activity-dependent central sensitization, lead revisions in cases with normal electrode impedance and no signs of local spread of the current seem not to be beneficial. It has to be recognized by physicians, so immediately began to reduce the stimulation current intensity.


Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics.Bacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa.Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa-associated otitis externa.Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for Validation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs.Our results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing. Pseudomonas aeruginosa is an important opportunistic pathogen of humans and animals, causing severe infections when the immune system is compromised [P. aeruginosa is a Gram-negative bacterium ubiquitously present in the water and soil. It is naturally resistant to penicillin and aminopenicillins, and to the first and second generations of cephalosporins. It is usually susceptible to aminoglycosides, fluoroquinolones, lipopeptides, ureidopenicillins, carboxypenicillins, carbapenems, and cephalosporins of the third generation [P. aeruginosa, however, can rapidly develop against any antibiotic [promised , 2, or apromised , 4. P. aneration . The restibiotic .Behavior of bacteria is coordinated, at the level of microbial community, by their gene expression at the intra-species level or less commonly between species. Such coordination of communal behavior is controlled through the production and detection of signal molecules or autoinducers, which is called quorum sensing (QS) –10. The Malassezia spp. (yeasts), and bacteria of the genera Staphylococcus, Proteus, Streptococcus and Pseudomonas, often cause complications to the inflammation of the ear canal  in dogs [P. aeruginosa in OE  increases the morbidity, prolongs the treatment, and reduces the possibility of a favorable outcome [P. aeruginosa to produce biofilms and synchronize its virulence through QS is very important for the conservation and/or perpetuation of infection [P. aeruginosa colonization and the subsequent activation of its QS [The secondary factors, including  in dogs , 18. Inv outcome , 20. Thenfection . The lonf its QS .P. aeruginosa QS system shows a hierarchical arrangement with las system controlling rhl [rhl system, where C4-HSL is the most significant autoinducing molecule [las system does not precede the activation of rhl, but just the opposite, indicating the environmental dependence of their hierarchy [P. aeruginosa AHL-dependent QS [P. aeruginosa genome is directly or indirectly regulated by this multi-signal QS system [las specific, some rhl, and others respond to both 3-oxo-C12-HSL and C4-HSL [P. aeruginosa QS network, being modulated in accordance to the environmental cues, also of host origin, influencing the virulence phenotypes of P. aeruginosa [One or several signal molecules may be involved in QS. In Gram-negative bacteria, the most common are N-acylhomoserine lactones (AHLs or AI-1) , 22, 23.ling rhl –8, 24–28ling rhl , 30. It molecule . In addimolecule  and a dimolecule , itself molecule  and contmolecule . Under sierarchy . There andent QS , 36 and S system . Some ged C4-HSL . In addiDetection of signal molecules associated with QS could serve as an indicator for the severity of infection, success of a treatment, and to estimate the outcome of disease . DisruptSeveral reports describing chemical analytical methods for the identification and quantification of selected AHLs are available. The use of a thin layer chromatography , 12; gasP. aeruginosa from canine OE clinical samples was introduced and validated. It consisted of the extraction of AHLs  from the ear rinses and their quantification using the liquid chromatography with tandem mass spectrometry (LC-MS/MS).In the present work, the analytical procedure for determination of AHLs of Solid standard substances C4-HSL, C6-HSL and 3-oxo-C12-HSL with certified purity of 97 %  were used. Stock standard solutions of individual AHLs with the concentration of 1 mg/ml  and 0.91 mg/ml (3-oxo-C12-HSL) were prepared in methanol. Mixed working standard solutions for preparing the calibration curve containing all the three selected AHL signal molecules, each at the concentration from approx. 1 to 60 ng/ml, were prepared in a mixture of methanol and deionized water (35 + 65). Dichloromethane used for the extraction of AHLs and methanol used for the mobile phase were of analytical and chromatography grade purity, respectively . As the mobile phase, a gradient mixing of methanol and deionized water containing 0.1 % formic acid (Merck) was used. For the rinsing of canine ear canals, saline solution (0.9 % NaCl) was employed.Samples were obtained by rinsing the ear canals of dogs with saline solution (30 ml). The collected samples were adjusted to the final volume of 30 ml by sterile saline, centrifuged at 3100 × g for 30 min at 4 °C, and the supernatants were stored at −80 °C till extraction.For validation, the ear rinses from healthy dogs spiked with known amounts of C4-HSL, C6-HSL and 3-oxo-C12-HSL were used. None of the samples contained AHLs in detectable concentrations prior to spiking. An aliquot of 0.320 ml of the mixed working standard solution containing AHLs in concentrations of approx. 60 ng/ml  was added to 10 ml of the supernatant, giving the final concentrations of approx. 2 ng/ml . Thus, a 10-ml sample contained approx. 20 ng of each AHL .PaOE dogs, collected weekly during the treatment, were used. Samples were taken during a scheduled veterinary checkup at the primary clinic or at the university clinic. As a control, the ear rinse from a healthy dog was employed.To test the validated analytical procedure with clinical samples, the ear rinses of two For the extraction of AHLs from the ear rinses, a procedure used for determination of AHLs in sputum , 12 was The particular AHLs were determined by LC-MS/MS at conditions described elsewhere , 45. In To perform the linearity test, and to determine the limit of detection (LOD) and the limit of quantification (LOQ), the mixed working standard solutions of AHLs at concentrations from approx. 1 to 60 ng/ml were used, corresponding to AHL concentrations of approx. 0.1 to 6 ng/ml in the ear rinses. The estimation of LOD and LOQ was based on visual evaluation, testing concentrations in the target range expected for AHLs in clinical samples. The LOQ estimate was defined as the lowest amount or concentration of an analyte that is reasonably achievable according to the target range and should be regarded as the reporting limit; LOD was estimated as one third (30 %) of LOQ .The repeatability of results was tested by analyzing five replicates of the ear rinse sample with the addition of C4-HSL, C6-HSL and 3-oxo-C12-HSL at 1.97 ng/ml  and 1.82 ng/ml (3-oxo-C12-HSL). The analyses were performed at the same day and by the same operator. For testing the within-laboratory reproducibility, five replicates of the sample were prepared for two more times by two operators at two different days. The results obtained from these experiments were also used for determination of the recovery.sr and sR, respectively) and relative standard deviation . The RSDR values were compared to the values calculated from the Horwitz equation (RSD = 2C)(1 − 0.5 log ), taken as a reference. The recovery was calculated according to the equation a represents the spiked concentration of the analyte in the sample [The mean e sample , 51.PaOE affected dogs with sterile saline, cotton-swab samples were taken from the ears for bacteriological examination. Samples were inoculated onto blood agar plates  supplemented with 5 % sheep blood and incubated aerobically at 37 °C for 48 ± 2 h. After 24-h and 48-h incubation, plates were examined for the growth of Pseudomonas spp. and other pathogenic bacteria. Colonies morphologically consistent with P. aeruginosa were subcultured on fresh blood agar plates for subsequent identification. Bacterial isolates were identified using the methods described by Quinn and colleagues [Prior to rinsing the ears of lleagues ; biochemIn Figs. y = 2027x + 708 (C4-HSL), y = 2603x + 2039 (C6-HSL) and y = 1594x + 204 (3-oxo-C12-HSL). Regression coefficients of the calibration curves were 0.999, 0.998 and 0.999, respectively. Based on the responses obtained, the lowest calibration point (1 ng/ml – or 0.1 ng/ml when referring to 10-ml volumes) was estimated as the lowest quantifiable concentration. Therefore, LOQ for 10-ml samples was estimated to 0.1 ng/ml (1 ng per 10-ml sample) and LOD accordingly lower to 0.03 ng/ml (0.3 ng per 10-ml sample). Expressed in molar concentrations, LOD and LOQ were approx. 175 pM and 584 pM for C4-HSL (with molar mass of 171.2 g/mol), 151 pM and 502 pM for C6-HSL (199.2 g/mol), and 101 pM and 336 pM for 3-oxo-C12-HSL (297.4 g/mol), respectively. Thus, with LOD in the range of 100–200 pM and LOQ in 300–600 pM, LC-MS/MS was confirmed suitable for the purpose as picomolar to micromolar concentrations were reported for clinical samples [Equations describing the calibration lines were 4 3-oxo-C-HSL. Reg samples , 48, 49.In Tables PaOE (positive samples) were analyzed. In Figs. PaOE diseased dog are shown, respectively. The positive-sample chromatogram clearly shows a peak belonging to C4-HSL  obtained from a healthy dog (negative control) and two dogs with HSL Fig. , which iPaOE collected at weekly intervals during the treatment  are given. The contents of C6-HSL and 3-oxo-C12-HSL were distinctly lower than of C4-HSL, making useful only the measurements of C4-HSL contents. In the first dog, the content of C6-HSL was much lower than of C4-HSL, but roughly correlated to fluctuations of C4-HSL; C6-HSL masses were close to LOQ , but also below LOQ (week 8) or even LOD . Surprisingly, the content of 3-oxo-C12-HSL was always below LOD. In the second dog, the contents of C6-HSL and 3-oxo-C12-HSL were below LOD but reached LOQ (C6-HSL) or LOD (3-oxo-C12-HSL) in the third and fourth week, which was in congruence with the increased C4-HSL levels.In Table P. aeruginosa strains. In the second dog, the disease management started with C4-HSL below LOD (day 0), but its content increased after the first week of treatment  and the reappearance of P. mirabilis. Later, in the second month of the treatment, the content of C4-HSL decreased and the secondary infection was resolved (week 6 and 8).In the first dog, the content of C4-HSL decreased in the first month of treatment, but then slightly increased in the sixth week and markedly in the eighth week of treatment , C6-HSL (22 %) and 3-oxo-C12-HSL (20 %) were compared to the reference values derived from the Horwitz equation: for the tested concentration of 2 ng/ml, the reference RSDR value was 41 % [RSDR values were below 41 %, the reproducibility was considered satisfactory.The detection and quantification limits of the analytical procedure were low enough  to enable determination of C4-HSL, C6-HSL and 3-oxo-C12-HSL in the ear rinses from dogs with bbs test , both bewas 41 % , 51. SinThe recoveries of C4-HSL, C6-HSL and 3-oxo-C12-HSL at the concentration of 2 ng/ml were 54, 89 and 79 %, respectively. Regarding requirements for the performance of analytical procedures , the reSample sediments, which were obtained after the initial centrifuging of the ear rinses, were tested for residual levels of AHLs prior to validation. This was performed to verify the predominating distribution of analytes into the liquid phase. Sediment analysis was the same as implemented for the supernatants. The quantities of individual AHLs were approximately 20-times lower in the sediments than in their respective supernatants (data not shown), indicating the residual level of less than 5 %. Therefore, only supernatants were used for further analysis.PaOE, which was confirmed with the routine bacteriological analyses. The treatment regimen was standard [PaOE diseased dogs with unlikely selection for bacterial resistance which would predispose the chronic character of the disease [Samples for the present study were collected from two dogs with standard –58 and i disease –58. A syPaOE, the content of C6-HSL was in general much lower than of C4-HSL, but the fluctuations of these two roughly correlated. The content of C6-HSL was approximating LOQ or even LOD. Moreover, the content of 3-oxo-C12-HSL was always below LOQ or, in most cases, below LOD. According to the results, only C4-HSL measurements showed applicable value. In the first dog, results showed that the content of C4-HSL decreased in the first month of Tris-EDTA/chlorhexidine treatment in accordance to expectations [P. aeruginosa strain.Regarding the total masses of AHLs in the ear lavages of dogs with ctations , but incP. mirabilis, a Gram-negative opportunistic pathogen ubiquitously present in the environment and a common secondary factor of OE in dogs [P. aeruginosa and P. mirabilis, since a variation of AHL QS system was observed in P. mirabilis with signaling molecules being structurally similar to AHLs [P. aeruginosa and of P. mirabilis, compounds capable of activating the AHL biosensor were found, not belonging to AHLs but to structurally similar diketopiperazines (DKPs). In concentration-dependent manner, DKPs were able to activate the AHL biosensor, although their physiological role remains to be discovered, but also suggesting the existence of cross talk among bacterial signaling systems. However, the QS system in Proteus sp. is not completely understood and its signaling molecules are not well defined. Although signaling molecules with the structure identical to AHLs are not produced by P. mirabilis [P. mirabilis reacts and changes the population features when AHLs produced by other Gram-negative bacteria are added to the culture media [P. mirabilis from the bacteriological sample. The marked increase in C4-HSL was detected in the third and fourth week, which coincided with an additional strain of P. aeruginosa and the reappearance of P. mirabilis in the bacteriological sample. After the fourth week, the C4-HSL content declined and P. mirabilis was no longer present. By the end of the treatment course, the additional strain of P. aeruginosa also disappeared.In the second dog, the content of C4-HSL was below LOD at the beginning of the disease management, but increased after the first week of treatment.  in dogs , 18, was to AHLs . Namely,irabilis , it was re media . The conPseudomonas infection.No AHLs were detected in the negative control sample from a healthy dog. The analytical procedure described and validated in this study is, therefore, capable of monitoring changes in AHL concentration throughout the treatment regimen, reflecting the state of i) 3-oxo-C12-HSL is not a major AHL present in the ear canals of PaOE-diseased dogs or in clinical samples in general; ii) deficiency of one or more P. aeruginosa QS components leading to reduced synthesis/deployment of 3-oxo-C12-HSL; iii) the in-vitro results, which are presently the gold standard for understanding the biology of QS and AHLs, are different from the in-vivo behavior of P. aeruginosa QS; and iv) limitations of the membrane transport, which are selective to effusion of 3-oxo-C12-HSL from P. aeruginosa cytoplasm.Concerning 3-oxo-C12-HSL, low concentrations were detected in this study. Similar was observed previously in samples from human patients with CF . Severalrhl system is ‘only’ subordinate to las, as we can speculate that rhl might be more adaptable to current conditions in its milieu or could support greater part of QS communication. A deficiency of one or more QS components of the rhl system was previously reported for P. aeruginosa canine OE isolates [P. aeruginosa mutants with the impaired las were also obtained from clinical samples [P. aeruginosa strains/isolates produce both C4-HSL and 3-oxo-C12-HSL [P. aeruginosa isolates from clinical samples or grown as biofilms when compared to those that are grown in laboratory as broth cultures in nutrient-rich media [P. aeruginosa is notorious for its ability to form biofilms [P. aeruginosa produces C4-HSL in excess of 3-oxo-C12-HSL when grown in biofilms but not in broth cultures [P. aeruginosa from the ear canals of PaOE-diseased dogs is also predominantly present as biofilms. Another reason for low concentrations of 3-oxo-C12-HSL in the studied clinical samples, in addition to QS deficiencies of P. aeruginosa strains and differential gene expression in biofilms, could be the fact that 3-oxo-C12-HSL requires an active membrane transport (pump) for its efflux from the cells to the surroundings, whereas C4-HSL diffusion has no such limitation and P. aeruginosa cells are freely permeable to C4-HSL [Regarding the results obtained in this study and previously for the samples from human CF patients , it is pisolates , and P.  samples . These Q samples . Not all-C12-HSL , 63. In ch media . Broth cch media , 12. P. biofilms . Isolatecultures . If the o C4-HSL , 66.P. aeruginosa is predominately growing in biofilms in the ear canals of PaOE-diseased dogs, another explanation for the low amounts of 3-oxo-C12-HSL can be suggested. Namely, AHLs are restricted in diffusion by the extracellular matrix of biofilms and they reach adequate intracellular concentrations for induction of QS-dependent genes at lower values in comparison to planktonic growth phenotype [rhl system, but more with planktonic phenotypes due to diffusion throughout the liquid milieu to reach concentrations high enough for the activation of rhl.If henotype . TherefoP. aeruginosa can adapt to different environments via its regulatory networks and multifunctional signal molecules [PaOE, or other P. aeruginosa infections, should have a beneficial impact on patients [olecules , 36, whipatients , 69. Plapatients , 71.P. aeruginosa and other bacteria. Detection and quantification of AHLs in clinical samples may provide an important diagnostic and prognostic tool to follow the disease progress, which has already been proven in human CF cases. It would also be possible to initiate and/or terminate, or evaluate, the success of antibiotic treatments in diseases of the PaOE complexity. In this study, the LC-MS/MS analytical procedure for determination of selected P. aeruginosa AHLs  in clinical samples obtained from PaOE-diseased dogs was introduced and successfully validated. For the clinical samples obtained from two PaOE-diseased dogs, low content of 3-oxo-C12-HSL was observed with C4-HSL predominating. To explain the phenomenon, it was suggested, in addition to other hypotheses, that P. aeruginosa is growing in the infected ear canals organized in biofilms.The QS system is a defining part of the pathologies caused by